Secretion of bacterial proteins is to be studied in vitro. In vitro enzyme synthesis will be conducted with cloned genes for secretory proteins including beta-lactamase from pBR322 and rbsP protein from E. coli. The gene for rbsP from lambda rbs will be cloned in pBR322. Genes for secretory proteins from B. subtilis will also be sought. Membrane vesicles from E. coli and other bacteria will be prepared and used to examine translocation of proteins synthesized in vitro. The energy requirement for translocation will be examined using appropriate mutants and energy poisons. Proteolytic processing of precursor forms will be studied using solubilized preparations from cell envelopes of E. coli and other bacteria.